Super-Resolution Imaging of Live Sperm Reveals Dynamic Changes of the Actin Cytoskeleton During Acrosomal Exocytosis Ana Romarowski1, Ángel G

RESEARCH ARTICLE Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis Ana Romarowski1, Ángel G. Velasco Félix2, Paulina Torres Rodrıgueź 3,Marıá G. Gervasi4, Xinran Xu5, Guillermina M. Luque1, Gastón Contreras-Jiménez3, Claudia Sánchez-Cárdenas3,Héctor V. Ramırez-Gó ́mez3, Diego Krapf5, Pablo E. Visconti4, Dario Krapf 6, Adán Guerrero2, Alberto Darszon3 and Mariano G. Buffone1,*

ABSTRACT 2015). The sperm AR occurs in mice in the upper segments of the Filamentous actin (F-actin) is a key factor in exocytosis in many cell female oviductal isthmus (Hino et al., 2016; La Spina et al., 2016; types. In mammalian sperm, acrosomal exocytosis (denoted the Muro et al., 2016) and is essential for the appropriate relocalization – acrosome reaction or AR), a special type of controlled secretion, is of proteins involved in sperm egg fusion (Satouh et al., 2012). regulated by multiple signaling pathways and the actin cytoskeleton. Acrosomal exocytosis is a highly controlled event that consists of However, the dynamic changes of the actin cytoskeleton in live sperm multiple stages that culminate in the membrane around the secretory are largely not understood. Here, we used the powerful properties of SiR- vesicle being incorporated into the plasma membrane. At the actin to examine actin dynamics in live mouse sperm at the onset of the molecular level, this complex exocytic process is regulated by AR. By using a combination of super-resolution microscopy techniques several signaling pathways involving: (1) membrane potential to image sperm loaded with SiR-actin or sperm from transgenic mice (De La Vega-Beltran et al., 2012), (2) proteins from the fusion containing Lifeact-EGFP, six regions containing F-actin within the sperm machinery system (Belmonte et al., 2016; De Blas et al., 2005), head were revealed. The proportion of sperm possessing these (3) small GTPases (Branham et al., 2009; Romarowski et al., 2015), 2+ structures changed upon capacitation. By performing live-cell imaging (4) changes in ion concentration, where Ca is a key player experiments, we report that dynamic changes of F-actin during the AR (Fukami et al., 2003; Romarowski et al., 2016a; Sánchez-Cárdenas occur in specific regions of the sperm head. While certain F-actin regions et al., 2014), and (5) changes in the actin cytoskeleton (Brener et al., undergo depolymerization prior to the initiation of the AR, others remain 2003; Romarowski et al., 2016b). In this regard, the expression of unaltered or are lost after exocytosis occurs. Our work emphasizes the actin and several actin-related proteins in mammalian sperm (some utility of live-cell nanoscopy, which will undoubtedly impact the search for of which are testis specific) suggests that actin polymerization and mechanisms that underlie basic sperm functions. depolymerization are important for sperm function (Gervasi et al., 2018; Romarowski et al., 2016b). This article has an associated First Person interview with the first author Filamentous actin (F-actin) is a key factor in exocytosis in many cell of the paper. types. It has been shown that F-actin holds together the molecular players of the secretory pathway, determines the precise site for granule KEY WORDS: Acrosomal exocytosis, Actin, Sperm exocytosis and shapes the exocytotic responses in secretory cells. In addition, stabilization of the actin network inhibits exocytosis, whereas INTRODUCTION depolymerization of this network increases the number of docked Exocytosis is the pathway by which cells selectively externalize secretory granules and enhances the exocytotic response (Chowdhury compounds and, as such, it is essential for a number of basic et al., 1999; Ehre et al., 2004; Gasman et al., 2004; Muallem et al., biological processes. In spermatozoa, acrosomal exocytosis (also 1995). As in somatic cells, the actin cytoskeleton of mammalian sperm known as acrosome reaction, denoted AR), a special type of is dynamic. While during sperm capacitation polymerization is controlled secretion, is essential for fertilization in mammals (Dan, predominant (Brener et al., 2003; Romarowski et al., 2015), during 1952). Sperm acquire the ability to undergo the AR in the female the AR, actin depolymerization occurs (Spungin et al., 1995). reproductive tract in a complex cascade of events all grouped under Previous studies regarding actin polymerization/depolymerization the name of capacitation (Puga Molina et al., 2018; Stival et al., were exclusively performed in fixed capacitated sperm or by means of using isolated membranes that had either been exposed or not to AR 1Instituto de Biologıá y Medicina Experimental (IBYME), Consejo Nacional de inducers (Brener et al., 2003; Romarowski et al., 2015). In addition, Investigaciones Cientıficaś y Técnicas (CONICET), Buenos Aires C1428ADN, most studies evaluating F-actin in mammalian sperm were performed Argentina. 2Laboratorio Nacional de Microscopıá Avanzada, Instituto de Biotecnologıa,́ Universidad Nacional Autónoma de México (UNAM), Cuernavaca, using phalloidin, which is toxic and not capable of crossing the cell Morelos 62210, México. 3Departamento de Genética del Desarrollo y Fisiologıá plasma membrane of live cells (Cooper, 1987; Vandekerckhove et al., Molecular, Instituto de Biotecnologıa,́ Universidad Nacional Autónoma de México 1985; Visegrády et al., 2004). Thus, the phalloidin staining approach is (UNAM), Cuernavaca, Morelos 62210, México. 4Department of Veterinary and Animal Science, Paige Labs, University of Massachusetts, Amherst, MA 01003, not suitable to study this dynamic process in real time using live cells. USA. 5Department of Electrical and Computer Engineering, School of Biomedical However, recent reports have demonstrated the use of a novel membrane Engineering, 1301 Campus Delivery, Fort Collins, CO 80523, USA. 6Instituto de permeable fluorescent probe, called SiR-actin, that binds to actin Biologıá Molecular y Celular de Rosario (CONICET-UNR), Rosario, Santa Fe ̌ S2000EZP, Argentina. filaments in vivo (Lukinavicius et al., 2014; Magliocca et al., 2017; Yamazaki et al., 2018). This probe allows one to image actin filaments *Author for correspondence ([email protected]) without the need to overexpress fluorescently tagged proteins, such as D.K., 0000-0002-2833-5553; M.G.B., 0000-0002-7163-6482 Lifeact, actin monomers or actin-binding proteins. In addition, SiR-actin provides a high signal-to-noise ratio, facilitating the visualization of the

Received 12 April 2018; Accepted 25 September 2018 finest details of the cortical actin network and it has been successfully Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958 visualized in neurons in stimulated emission depletion (STED) produced as a modified form of jasplakinolide, a drug that super-resolution microscopy studies (D’Este et al., 2015). is normally used to stabilize polymerized actin in vitro,itis By using this powerful new probe, we aimed to examine actin recommended to minimize the concentration of the probe and dynamics in live mouse sperm, specifically during the AR process. ideally not have a concentration of >100 nM, since higher We hypothesize that a complex and highly regulated process, such as concentrations could affect the actin cytoskeleton dynamics the AR, will be accompanied by specific changes in actin dynamics. (Lukinaviciuš et al., 2014). For this reason, we incubated sperm To this end, in live-cell imaging experiments, we set up the conditions with increasing concentrations of SiR-actin (50 to 500 nM). to simultaneously monitor actin cytoskeleton remodeling and AR Although 500 nM of SiR-actin resulted in a bright fluorescent occurrence by monitoring SiR-actin and FM4-64, respectively (Mata- signal in the sperm head and flagellum, we found that 100 nM SiR- Martínez et al., 2018; Romarowski et al., 2016a; Sánchez-Cárdenas actin (in accordance with the manufacturer’s recommendations to et al., 2014). For the first time, we report the observation of acrosomal avoid artifacts) showed a good signal-to-noise ratio (Fig. 1A). The exocytosis via super-resolution microscopy in real time. Our work signal observed using 50 nM was barely detectable. As a control, provides a detailed characterization of the F-actin cytoskeleton in cells that were not loaded with SiR-actin displayed no fluorescence the sperm head by using SiR-actin in combination with different (auto-fluorescence was not significant when excited by the far-red super-resolution microscopy approaches and transgenic mice wavelength used to detect SiR-actin; data not shown). Analysis of containing the F-actin-binding protein Lifeact-EGFP. Additionally, sperm motility by computer-assisted sperm analysis (CASA) was we observed dynamic changes of F-actin in restricted regions of the used to evaluate whether 100 nM of SiR-actin affected sperm sperm head prior to and after the initiation of the AR. Finally, our motility and viability. As shown in Fig. S1, this probe concentration work emphasizes the advantage of live-cell nanoscopy for the study did not alter either the viability or sperm motility. of ultra-structures in sperm as well as in other cell types. These F-actin is normally visualized in fixed mammalian sperm by imaging capabilities will undoubtedly impact the study of using fluorophore-conjugated phalloidin. To compare the staining mechanisms that underlie basic sperm functions. produced by phalloidin and SiR-actin, we loaded the cells with both probes. By using confocal microscopy, we found that unlike RESULTS phalloidin, which stains the sperm head homogenously, SiR-actin SiR-actin, a new versatile tool to visualize F-actin in shows F-actin structures in specific regions of the sperm mouse sperm head (Fig. 1B). To study the organization of F-actin in mouse sperm, we exploited Previous studies from several laboratories report an increase in the membrane permeability of SiR-actin. Because SiR-actin is actin polymerization during capacitation in both the head and the

Fig. 1. SiR-actin, a novel method to visualize F-actin in mature mouse sperm. (A) Representative images of sperm loaded with different SiR-actin concentrations (50–500 nM). Fluorescence intensities are represented as a color map, see reference bar on top of panel A; weak (–) to strong (+) fluorescence. (B) Representative images of sperm labeled with both SiR-actin and Alexa-Fluor-488–phalloidin. The image on the right is a magnification of the boxed area in the merged picture. n=3. (C) Representative images obtained by image-based flow cytometry for non- capacitated (NC) and capacitated (CAP) sperm showing the bright field (BF), the PI and the SiR-actin channels. (D) Box plot of a representative experiment comparing NC and CAP sperm. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show 1.5 times the interquartile range; the notches represent the 95% confidence interval around the median. Statistical analysis was performed using a Kolmogórov-Smirnov test. *P<0.05. AUF, arbitrary units of fluorescence. A summary of all experiments can be seen in Fig. S2E, n=12 mice. (E) Representative images of SiR-actin loaded sperm before and after 60 min incubation under CAP and NC conditions (n=3). Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958 tail of mouse sperm. To further investigate whether SiR-actin can phalloidin, we aimed to visualize these structures in sperm from also reveal F-actin formation during sperm capacitation, cells were transgenic Lifeact-EGFP mice (Riedl et al., 2010). Lifeact is a loaded with SiR-actin and incubated under capacitating and 17-amino-acid peptide, which stains F-actin structures in eukaryotic non-capacitating conditions, and analyzed via image-based flow cells and tissues, which does not interfere with actin dynamics cytometry. The settings used to perform these experiments are in vitro and in vivo. shown in Fig. S2. This analysis confirmed previous observations Lifeact-EGFP sperm displayed a strong fluorescent signal in the obtained using phalloidin. The fluorescence intensity of sperm head and in the tail. The fluorescent signal in the flagellum is mostly incubated under capacitating conditions was significantly higher from the mid-piece, but F-actin is also visible in the principal piece. than in the non-capacitated cells, indicating actin polymerization Using sperm from Lifeact-EGFP transgenic mice, we observed during this process (Fig. 1C,D; Fig. S2E). Similar results were F-actin structures in the sperm head with a similar localization obtained using confocal microscopy (Fig. 1E). to that seen in SiR-actin loaded sperm (Fig. 3A). These results suggest that the novel F-actin structures revealed by SiR-actin are SiR-actin reveals novel F-actin structures in the sperm head not artifacts. Using confocal microscopy, we found six different F-actin structures within the sperm head as follows: (1) the perforatorium, F-actin localization in the sperm head using STORM super- staining in the more apical tip of the head; (2) the lower acrosome, resolution microscopy labeling in the acrosomal region close to the anterior tip of the head; To investigate and further corroborate the organization of F-actin in (3) the upper acrosome, staining over the entire cortical acrosomal the sperm head in more detail, we used the super-resolution imaging cap; (4) a ventral, fluorescent signal in the lower edge of the head; technique stochastic optical reconstruction microscopy (STORM), (5) the septum, a clear fluorescent boundary between the equatorial which has previously been used to visualize flagellar proteins in and post-acrosomal segment; (6) a neck, a focused signal in the neck mouse sperm, such as CatSper proteins (Chung et al., 2014, 2017; region. These patterns are represented in the schematic diagram Gervasi et al., 2018). We utilized this approach to study the shown in Fig. 2A. distribution of F-actin in the sperm head. Because it is not possible When we compared the percentage of sperm that displayed each to use STORM with SiR-actin, owing to its optical properties, we of these patterns under capacitating vs non-capacitating conditions, used phalloidin–Alexa-Fluor-647-stained sperm and recorded a no significant differences were observed (Fig. 2B). To further very similar F-actin distribution to that observed with the SiR-actin characterize the distribution of these patterns in the whole probe (Fig. 3B). This result further validates the novel F-actin population, we analyzed whether they were correlated in non- structures in the sperm head revealed by SiR-actin. Also, these capacitated and capacitated sperm, using a correlation matrix of the experiments demonstrated the advantage of using super-resolution six patterns defined above (Fig. 2C). A positive correlation microscopy to reveal F-actin structures in the sperm head that coefficient would thus imply the structures are more likely to be otherwise displayed a homogeneous distribution due to the present together and a negative correlation would imply one diffraction-limited acquisition of these images. structure is more likely to be present in the absence of the second To visualize the three-dimensional F-actin distribution in the one. In non-capacitated sperm, the pairs of patterns upper sperm head, the molecular localizations found by STORM in xyz acrosome/septum, septum/neck and neck/lower acrosome were coordinates were used to build z stacks of 2D x-y reconstructions, significantly positively correlated. In contrast, the ventral region which were later rendered into a 3D image through the 3D viewer was negatively correlated with the neck. Strikingly, all these plugin in ImageJ. 3D-reconstructed videos can be observed in correlations disappeared upon sperm capacitation, revealing Movies 1 and 2. changes undertaken by the actin cytoskeleton. In capacitated sperm, no significantly positively correlated patterns were Super-resolution microscopy of F-actin and plasma observed and the negatively correlated pairs were ventral/ membrane using SiR-actin in combination with FM4-64 perforatorium and upper acrosome/lower acrosome. To obtain sub-diffraction images of the F-actin network using As we observed great heterogeneity in the sperm population, with SiR-actin, we employed a recently developed variation of the most of the cells displaying two or more patterns simultaneously, we localization technique, the Bayesian analysis of Blinking and classified the cells using a six-position binary code developed Bleaching (3B), which allows analysis of data with multiple specifically for this purpose (Fig. 2A). The binary code was defined overlapping fluorophores in every image (Cox et al., 2012). Cells as 1 or 0 (presence or absence of signal at a given region, loaded with SiR-actin were also stained with the plasma membrane respectively). In addition, each position in the 6-digit code dye FM4-64 used for visualization of exocytosis and endocytosis represents one of the patterns. Using this code, we evaluated the (Romarowski et al., 2016a; Sánchez-Cárdenas et al., 2014). proportion of each combination of patterns in non-capacitated and The images were obtained via TIRF microscopy (Fig. 4A,B) capacitated sperm and found that five of them were significantly followed by 3B reconstruction (Fig. 4C). The areas delimited in different between these two conditions (Fig. 2D). In the chart of Fig. 4C are shown in higher magnification in Fig. 4D–G. As Fig. 2E, the five pattern combinations that increase or decrease shown in Fig. 4D and F, by applying this reconstruction algorithm, during capacitation can be observed. Those five patterns are shown it is possible to observe in great detail the cortical actin structure in the representative pictures of Fig. 2F. The arrows indicate the (green) underneath the plasma membrane (magenta) in both increase or decrease of these patterns during capacitation (pointing the acrosomal area and the neck-midpiece region. In addition, the upward or downward, respectively) (Fig. 2F). perforatorium region, which is localized at the very tip of the sperm head, possessed a well-defined F-actin structure whose Similar F-actin structures are observed in transgenic function is unknown (Fig. 4E). Fig. 4G shows the F-actin structure Lifeact-EGFP mice localized at the boundary between the equatorial region and Because SiR-actin revealed novel F-actin structures in the sperm the post-acrosomal area (previously called ‘septum’) (see also head that are not observed when staining via the widely used Fig. 2A). Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

Fig. 2. SiR-actin reveals novel F-actin structures in the sperm head. (A) Representative diagram showing the six different F-actin structures within the sperm head that we denote as the perforatorium, lower acrosome, upper acrosome, ventral, septum and neck. A representative confocal image of each pattern is displayed. A binary code was used to determine the presence of these patterns in all the individual cells assessed. The binary code was defined as 1 (presence of signal at a given region) or 0 (absence of fluorescent signal). In addition, each position in the 6-digit code represents one of the patterns: the positions (from left to right) are assigned to perforatorium, upper acrosome, neck, septum, ventral and lower acrosome. (B) The proportion of each individual pattern in NC and CAP sperm was evaluated. Dots represent the mean from each mouse; the blue lines represent the median and the red lines are the s.d. from all the experiments conducted. Student’s t-test was performed; n=3, analyzed cells=600. The P values originated in this statistical comparison were: 0.49 for perforatorium, 0.076 for upper acrosome, 0.1 for neck, 0.99 for septum, 0.99 for ventral and 0.34 for lower acrosome. (C) Correlation matrix of the six patterns in non-capacitated (NC) and capacitated (CAP) sperm. The hypothesis that the correlation coefficients (correlation matrix elements) were significantly different than zero was tested. To facilitate the observation of significant correlations, all the matrix elements without at least 5% correlation significance were set to zero. In NC sperm, the positively correlated pairs had P-values of <0.003, while the negative correlation had P<0.0005. In CAP sperm, the negative correlation of ventral/perforatorium and upper acrosome/lower acrosome had P<0.02 and P=0.0006, respectively. Student’s t-test was performed; n=3, 221 non-capacitated sperm and 342 capacitated sperm were analyzed. (D) By using the binary code, the proportion of each pattern combination in NC and CAP sperm was evaluated. Only five pattern combinations are significantly different between these two conditions (those that are below the red line representing P<0.05). The P-values originated from this statistical comparison are represented in the y-axis of the graph. In the x-axis, each individual pattern combination is represented. Fisher’s test was performed; n=3, analyzed cells=600. (E,F) Pie chart (E) showing the five pattern combinations that increase or decrease their proportion during capacitation. Those five pattern combinations are shown in the representative pictures (F). The arrows indicate the increase or decrease of these pattern combinations during capacitation (pointing upward or downward, respectively).

Highlighting the powerful capabilities of using super-resolution (Fig. S3). The well-defined F-actin structure observed prior to microscopy to study these processes, we observed changes in the capacitation is altered, resulting in a more diffuse staining over that F-actin network in or surrounding the perforatorium area. When area. These type of observations cannot be visualized using we compared the super-resolution images of non-capacitated conventional confocal microscopy alone, indicating that there is and capacitated sperm, we found that the perforatorium F-actin active actin remodeling in this structure during the capacitation cytoskeleton undergoes structural modifications during capacitation process. Therefore, these results highlight the powerful capabilities Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

of this event. Live sperm loaded with SiR-actin and FM4-64 were immobilized in concanavalin A-coated slides as previously described (Hirohashi et al., 2015; Romarowski et al., 2016a), to continuously monitor changes in SiR-actin in those cells that undergo exocytosis (as judged by the increase in the FM4-64 fluorescence). An agonist of the AR, such as the Ca2+ ionophore ionomycin, was added to trigger the initiation of the AR. Cells were recorded at 37°C for 20 min, as described in the corresponding Materials and Methods section. Following acquisition, the images were reconstructed using 3B analysis. Our observations reveal that, among the six F-actin structures in the head that were described above, three of them change during AR: (1) the septum, (2) the lower acrosome and (3) the upper acrosome. Interestingly, the changes in these regions occurred with different kinetic behaviors as below.

Septum Fig. 5A shows a representative super-resolution image of a sperm that, at the beginning of the recording, possesses the septum F-actin structure previously described (left). The time course sequence of that structure after the addition of ionomycin is shown at higher magnification. The regions of interest (ROIs) corresponding to the septum region (for SiR-actin) and the whole sperm head (for FM4-64) (Fig. 5B) are shown together with the corresponding fluorescence traces (Fig. 5C). The fluorescence curves indicate that F-actin depolymerization in the septum area preceded the AR initiation by ∼100 s (Fig. 5C). Fig. S4A–C illustrates a representative sequence of images of a sperm that did not initiate the AR and conserved the F-actin structure in that region. A video associated with this cell that loses the septum F-actin structure can be observed in the Movie 3.

Lower acrosome This region also depolymerized before the initiation of the AR (see Fig. 5D). Fig. 5D displays a representative super-resolution sequence of images of a sperm that underwent actin depolymerization in this region after stimulation with ionomycin, prior to AR initiation. The ROIs corresponding to the lower segment region (for SiR-actin) and the whole sperm head (for FM4-64) are Fig. 3. F-actin visualization in the sperm head using Lifeact-EGFP shown together with the corresponding fluorescence traces (Fig. 5E transgenic sperm, and STORM super-resolution microscopy using and F, respectively). These curves show that the depolymerization in phalloidin. (A) Non-capacitated sperm from Lifeact-EGFP mice displayed the lower acrosome area also preceded the initiation of the AR by similar F-actin structures as observed with SiR-actin. Lifeact-EGFP sperm ∼50 s (Fig. 5F). A representative sequence of images of a sperm that displayed a strong fluorescent signal in the head and in the tail (i). The fluorescent signal in the flagellum is mostly from the mid-piece, but F-actin is did not initiate the AR and conserved the F-actin structure in the also visible in the principal piece. The arrows indicate different structures also lower acrosomal region is shown in Fig. S4D–F. A video associated observed when using SiR-actin, such as the perforatorium (ii), ventral and with this cell that loses the lower acrosome F-actin structure can be upper acrosome (iii) and septum (iv). BF, bright field. (n=3). (B) Representative observed in Movie 4. images of 3D STORM super-resolution microscopy of F-actin in mouse sperm. Scale bar: 500 nm. Non-capacitated sperm were stained with Alexa- Upper acrosome Fluor-647–phalloidin. Six representative images revealing the distribution of F-actin in the sperm head are shown (n=13). The 3D-reconstruction is This region also displayed a loss of SiR-actin fluorescence intensity colored according to the localization height as indicated in the color map. during the AR. As shown in Fig. 6B, the F-actin network that The 3D-reconstruction videos can be observed in Movies 1 and 2. resembles the subcortical F-actin localization (also shown in Fig. 4D), is disassembled during the AR. However, analysis of the ROI corresponding to the upper acrosome area (Fig. 6A) and the of super-resolution microscopy to visualize cytoskeletal structures resulting fluorescent traces (Fig. 6C) revealed that F-actin loss in the mouse sperm head. occurred ∼400 s after AR initiation, as judged by the increase in FM4-64 fluorescence. Similar to what was observed in the other two Dynamic changes of F-actin during acrosomal exocytosis regions that displayed depolymerization during the AR, sperm that occur in specific regions of the sperm head did not initiate the AR conserved the F-actin structure in the upper In the following experiments, we aimed to visualize dynamic acrosomal region (Fig. S4G–I). changes of the actin cytoskeleton during the AR, with emphasis on The decrease in SiR-actin fluorescence observed in the upper observing the depolymerization of the F-actin structure at the onset acrosome occurred several seconds after initiation of the AR, Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

Fig. 4. Visualization of F-actin and plasma membrane in mouse sperm by super-resolution microscopy. (A,B) Diffraction-limited images obtained by TIRF microscopy of a sperm loaded with FM4-64 (A) and SiR-actin (B). (C) To obtain sub-diffraction resolution of F-actin using SiR-actin, we employed 3B analysis. The 3B super-resolution reconstruction of the sperm shown in A and B, after merging the two fluorescence channels. Scale bar: 1 μm. F-actin (revealed by SiR-actin) is shown in green and the plasma membrane (stained with FM4-64) in magenta. The areas depicted in C are shown in higher magnification in D–G. (D) Higher magnification of the area (i) showing the cortical F-actin network in close association with the plasma membrane. (E) Higher magnification of the area (ii) showing the F-actin localized in the perforatorium. (F) Higher magnification of the area (iii) showing F-actin present in the sperm neck. (G) Higher magnification of the area (iv) showing the septum F-actin structure.

suggesting that F-actin loss may occur as a result of membrane of the recording possessed the F-actin structure corresponding to the vesiculation, and that release of the protein is caused by the AR. perforatorium (Fig. 7A–C), ventral or neck region (Fig. 7D–F). The Supporting this hypothesis, super-resolution analysis of sperm that ROIs corresponding to those regions (for SiR-actin) and the whole initiated the AR revealed that cortical actin remained unaltered sperm head (for FM4-64) are shown (Fig. 7A, and D), together with during the initial steps of membrane vesiculation. The time course the corresponding fluorescence traces (Fig. 7C and F). These traces of the formation of a hybrid vesicle during AR initiation is show that depolymerization did not occur before or after the illustrated in Fig. 6D,E. A clear decrease in FM4-64 fluorescence initiation of the AR. and SiR-actin is observed as a result of membrane loss (see right arrow in panel 109.8 s). The membrane lost in that region is DISCUSSION observed in the newly formed vesicle together with the F-actin that Once capacitation takes place, mammalian sperm are ready for a left the cortical actin in the sperm head (see left arrow in panel regulated AR. Although the identity of the physiological AR 109.8 s). inducer in vivo is controversial (Buffone et al., 2014), most authors To further validate our results, we analyzed the fate of these three agree on postulating Ca2+ elevation as one of the initial events F-actin structures during the AR induced with another agonist, leading to exocytosis. However, between the rise of intracellular progesterone. Results obtained with progesterone were overall Ca2+ and exocytosis, there is a lag period indicating that exocytosis similar to those observed with Ca2+ ionophore; sperm undergoing in sperm relies on other signaling pathways downstream of the the AR displayed loss of SiR-actin fluorescence in the septum, and increase in Ca2+ (Romarowski et al., 2016a). Among them, we can lower and upper acrosome regions (Fig. S5). However, their kinetics highlight the involvement of phospholipases (Cohen et al., 2004; were slightly different. The loss of F-actin in the septum and lower Fukami et al., 2001), cAMP (Branham et al., 2006; De Jonge et al., acrosome occurred ∼400 s and ∼40 s before the initiation of the AR, 1991; Lucchesi et al., 2016), exchange factors (i.e. EPAC) respectively. In the case of the upper acrosome, we observed that the (Branham et al., 2009), Rab3A and SNARE proteins (De Blas loss of SiR-actin fluorescence occurred at about the same time of the et al., 2005; Quevedo et al., 2016), small GTPases (Baltiérrez- FM4-64 fluorescence increase. The other regions containing F-actin Hoyos et al., 2012; Belmonte et al., 2016; Fiedler et al., 2008; (ventral, perforatorium and neck) did not change in sperm Pelletán et al., 2015; Romarowski et al., 2015) and undergoing exocytosis (Fig. S6). The differences observed may depolymerization of the actin cytoskeleton formed during be related to distinct mechanisms that these two agonists drive to capacitation (Brener et al., 2003; Spungin et al., 1995). In this stimulate the AR. paper, we focused on observing specific dynamic changes of the F-actin network at the onset of the AR. Here, we showed actin Specific F-actin structures in the sperm head did not depolymerization in specific regions of the sperm head during the change as a consequence of the AR AR, an event that was not observed before owing to technological While three specific regions (septum, lower acrosome and upper limitations. Our approach used super-resolution microscopy in live acrosome) showed actin depolymerization or loss accompanying the cells where specific changes in F-actin are monitored in real time at AR, the other three F-actin structures in the head (ventral, the onset of the AR. perforatorium and neck) remained unchanged even though sperm We used SiR-actin, a recently developed probe, to monitor underwent AR. Fig. 7 shows a representative sequence of images of F-actin dynamics in live cells (D’Este et al., 2015). The ability to use sperm that were stimulated with ionomycin and that at the beginning live sperm in our experiments as opposed to the use of traditional Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

Fig. 5. See next page for legend. phalloidin staining in fixed cells, revealed detailed spatio-temporal allowing detailed spatial localization of the actin network. We information about F-actin. These observations were consistent with observed six different regions within the sperm head that are similar results obtained in sperm from transgenic mice containing enriched in polymerized actin. One of them is the perforatorium, a Lifeact-EGFP, and with data from super-resolution methods prominent feature of falciform-shaped sperm heads, such as those

[STORM, super-resolution radiality fluctuations (SRRF) and 3B] found in mice. The perforatorium in mice has the shape of a curved Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

Fig. 5. Specific dynamic changes in the actin cytoskeleton before for perinuclear theca organization and sperm plasma membrane acrosomal exocytosis. Sperm were loaded with SiR-actin (green) and attachment to those underlying structures (Selvaraj et al., 2009). Our FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging. studies revealed the presence of F-actin in this region that remains Following acquisition, images were analyzed using 3B analysis. (A) Representative super-resolution image of a sperm that, at the beginning of unaltered during the AR. the recording, possesses the septum F-actin structure previously described We also found that the proportion of sperm with a pattern that (left). In higher magnification, the time course sequence of that structure after includes the perforatorium F-actin structure increased with addition of ionomycin (black arrow) is shown. The F-actin structure in the capacitation (Fig. 2). Interestingly, super-resolution analysis of the septum region depolymerizes prior to the initiation of the AR. ″ represents time F-actin network in this region showed subtle differences between in seconds. The white arrows highlight the F-actin structure in the septum non-capacitated and capacitated sperm (Fig. S3). The F-actin region. (B,C) The ROIs corresponding to the septum region (for SiR-actin) and the whole sperm head (for FM4-64) (B) are shown together with the structure in capacitated sperm looked more diffuse compared to the corresponding fluorescence traces (C). The corresponding movie for this well-defined structure in non-capacitated cells, suggesting that actin experiment is shown in Movie 3. (D) Representative super-resolution images of reorganization took place in that specific region during capacitation. a sperm that undergoes actin depolymerization in the lower acrosome region Additional experiments will be necessary to understand the after stimulation with 10 µM of ionomycin prior to the initiation of the AR. implication of this change in the mechanisms related to the AR (E,F) The ROIs corresponding to the lower acrosome region (for SiR-actin) and and fertilization. However, because we previously observed that the whole sperm head (for FM4-64) (E) are shown together with the most sperm that undergo the AR initiate the Ca2+ increase in corresponding fluorescence traces (F). The corresponding movie for this experiment is shown in Movie 4 (n=6; 62 cells analyzed). that region of the cell (Romarowski et al., 2016a), we speculate that changes in the actin network may modulate the Ca2+ wave associated triangular rod at the apex of the sperm head, and splits into three with the AR. In this regard, the influence of the cytoskeleton in Ca2+ interconnected prongs, one dorsal and two ventral, as it passes over wave propagation and permeability has been previously reported the nucleus (Clermont et al., 1990). Although the function of sperm in other systems (Torregrosa-Hetland et al., 2011). ultrastructural elements are poorly understood, it is thought that they In somatic cells, membrane-associated cytoskeletal components play a mechanical role during egg penetration in fertilization have been implicated in organizing membrane domains by (Korley et al., 1997). The presence of cytoskeletal structures in this providing lipid and protein tethers within the membrane (Suzuki sperm region, which is mostly occupied by the perinuclear theca, et al., 2017). Another F-actin structure revealed by SiR-actin is has been documented previously (Clermont et al., 1990). Besides localized at the boundary between the equatorial segment and the these cytoskeletal structures, the components of this region are post-acrosomal region. Previous studies have demonstrated the largely unknown (Korley et al., 1997; Oko and Morales, 1994). presence of a physical barrier in that region that impeded normal Protein FABP9 has been invoked as being a critical building block lipid diffusion (Jones et al., 2007). This physical barrier in sperm is

Fig. 6. Specific dynamic changes in the actin cytoskeleton after initiation of acrosomal exocytosis. (A) Sperm labeled with SiR-actin (green) and FM4-64 (magenta). The ROIs used for the analysis, corresponding to the upper acrosome region (for SiR-actin) and the whole sperm head (for FM4-64), are depicted. (B) Representative sequence of images of a sperm loaded with SiR-actin and FM4-64 that displayed a decrease in SiR-actin fluorescence in the upper acrosome region after stimulation with 10 µM ionomycin (addition indicated by black arrow). ″ represents time in seconds. (C) The resulting fluorescence traces revealed that the loss of F-actin occurred after the initiation of the AR as judged by the increase in FM4-64 fluorescence (n=6; 62 cells analyzed). (D,E) Super-resolution analysis of a sperm loaded with SiR-actin and FM4-64 showing the formation of a hybrid vesicle at the onset of the AR. The area depicted in D is shown at a higher magnification in E, at different times after 10 µM ionomycin addition. The cortical actin remained unaltered during the initial steps of membrane vesiculation. The arrows in the panel at 109.8 s indicate a clear decrease in FM4-64 fluorescence and SiR-actin as well as the newly formed vesicle containing both F-actin and plasma membrane. See Movie 5. Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

Fig. 7. Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with ionomycin (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) (n=6. 62 cells analyzed). composed of an electron-dense matrix, forming a demarcation line movement. Our hypothesis is that actin depolymerization is between both compartments (Selvaraj et al., 2009), which is essential for facilitating the migration of IZUMO1 to the compatible with the existence of a dense cytoskeletal structure. equatorial domain. Interestingly, the distribution of the sperm- However, the presence of F-actin in this region has never been specific plus-end actin-capping protein CAPZA3 shows a dynamic clearly demonstrated and visualized in live cells. Here, we showed pattern of localization (Sosnik et al., 2010). CAPZA3 is completely the existence of an F-actin structure and, in addition, we provided relocated to the post-acrosomal region when IZUMO1 has only evidence of its dynamic behavior prior to the initiation of the AR. started to redistribute, suggesting that the redistribution of these two We have observed that the F-actin network in this boundary is proteins might occur through different mechanisms. In this new depolymerized before the AR begins. We hypothesize that this location, CAPZA3 could play a role in stabilizing polymerized actin structure has a function related to the preparation undertaken by the and a subsequent myosin-dependent movement of IZUMO1 to the sperm for fusion with the oocyte. In this regard, IZUMO1 is the only fusogenic site using the actin cytoskeleton. sperm protein with a demonstrated role in sperm–egg fusion (Inoue The other regions that also displayed an active depolymerization et al., 2005). It was previously observed that IZUMO1 localizes to during AR were the lower and upper acrosome regions. the anterior acrosome in intact sperm, but once the the AR is We observed that the signal in the acrosomal region that is close initiated, it moves to the equatorial peri-acrosomal segment, the to the anterior tip of the head is lost in all cells that undergo AR. region by which this cell binds and fuses with the egg oolema Similar to what occurs with the septum, we also observed that this (Miranda et al., 2009; Sosnik et al., 2009). Sperm from TSSK6-null depolymerization occurs prior to the initiation of membrane fusion mice that are deficient in IZUMO1 movement cannot fuse with eggs (∼50 s) in all cells analyzed. In contrast, the F-actin network present and display abnormalities in actin polymerization (Sosnik et al., in the upper acrosome (for cells that displayed a cortical staining 2009). In addition, IZUMO1 re-localization is blocked in the over the entire acrosomal cap that resembles the subcortical presence of inhibitors of actin dynamics, such as cytochalasin D, F-actin localization) is lost during or after the initiation of the latrunculin A and the myosin kinase blocker blebbistatin (Sosnik AR, as judged by the increase in FM4-64 fluorescence. Our data et al., 2009), even in sperm that undergo the AR. Our work, together suggest that, although this loss in SiR-actin fluorescence may be with others (Sosnik et al., 2009; Zhou et al., 2017) suggests indicative of depolymerization, it may also occur as a result of that exocytosis completion can be dissociated from IZUMO1 membrane loss caused by the AR. Supporting this latter possibility, Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958 super-resolution analysis revealed that the cortical actin remained further understand the process of fertilization. We expect that these unaltered during the initial steps of membrane vesiculation, novel real-time super-resolution methodologies will be useful in although this observation requires further investigation (Fig. 6 and investigating the spatial and temporal relationship between the Movie 5). Interestingly, the proportion of sperm with a pattern increase in Ca2+ and actin dynamics during the AR. In addition, this containing the septum or the lower acrosome F-actin structure also approach can also be adapted to other cell types where live imaging increased with capacitation (Fig. 2E,F). Those patterns are the ones of the actin cytoskeleton is required. SiR-actin provides many that are depolymerized with AR induction prior to its initiation. advantages compared to other methods: (1) it is easy to use, (2) it is Finally, in somatic cells, actin depolymerization occurs as a result membrane permeable, (3) it can be used in live cells without any of the activation of Ca2+-dependent actin-severing proteins, such as fixation, and (4) its far-red absorption and emission wavelengths gelsolin, cofilin and scinderin, which have been described in human, does not overlap with other commonly used fluorophores. On the mouse and guinea pig sperm (Finkelstein et al., 2010; Pelletier et al., other hand, the only potential disadvantage to be considered is 1999; Romarowski et al., 2015). In particular, Ca2+-dependent the possible alteration of actin dynamics, although it is reported activation of gelsolin has been proposed to lead to the AR (Finkelstein that this possibility does not occur or is minimized by using low et al., 2010). Consistent with this, our preliminary data indicate that concentrations of the probe (Melak et al., 2017). actin depolymerization follows an initial increase in intracellular Ca2+ and precedes fusion between the outer acrosomal membrane and the MATERIALS AND METHODS plasma membrane. We have also reported, in a single-cell analysis, Reagents that a specific transitory increase in intracellular Ca2+ triggers the AR All chemicals were purchased from Sigma-Aldrich Chemical Co. (St Louis, (Romarowski et al., 2016a). Despite these observations, little is MO) unless stated otherwise. SiR-actin was obtained from Cytoskeleton – – known about the relationship between Ca2+, actin dynamics and the (Denver, CO). FM4-64, Alexa-Fluor-488 phalloidin and Alexa-Fluor-647 phalloidin were from Thermo Fisher Scientific (Waltham, MA). Ionomycin different membranes that conform the sperm apical head. was from Alomone Laboratories (Jerusalem, Israel). In summary, we have simultaneously visualized actin dynamics and AR in live sperm by super-resolution microscopy. Based on Animals our observations, we believe that in a specialized cell, like the CD1 and Lifeact-EGFP (Riedl et al., 2010) mature (10–12 weeks old) mammalian sperm, actin depolymerization (septum, lower male mice were used in this work. Animals were maintained at 23°C with a acrosome) and actin reorganization (perforatorium) in specific 12-h-light–12-h-dark cycle. Animal experimental procedures were reviewed regions of the cell coexist. These observations are summarized in and approved by the Ethical Committee of Instituto de Biotecnologia/ the schematic model represented in Fig. 8; when capacitated sperm UNAM and of the Instituto de Biologia y Medicina Experimental and in are exposed to stimuli that promote AR, actin depolymerization strict accordance with the Animal Care and Use Committee (IACUC) occurs in two specific regions of the sperm head, prior to the guidelines of University of Massachusetts, Amherst and Colorado State initiation of the AR, the lower acrosome and the septum. Following University and the Guide for Care and Use of Laboratory Animals approved by the National Institutes of Health (NIH). the initiation of the AR, the cortical F-actin (upper acrosome) is lost as a result of the formation of hybrid vesicles and not as a result of Sperm capacitation active depolymerization. This novel approach of using SiR-actin In all the experiments, cauda epididymal mouse sperm were collected from opens new avenues to integrate the two events of the AR and actin retired male breeders by placing minced cauda epididymis in a modified dynamics to other molecular processes associated with exocytosis Krebs–Ringer medium (Whitten’s-HEPES-buffered medium; WH) as (i.e. changes in Ca2+ and membrane potential, etc.) in order to previously described (Romarowski et al., 2015). The capacitating medium

Fig. 8. Schematic diagram of the progression of acrosomal exocytosis. When capacitated sperm are exposed to stimuli that promote AR, actin depolymerization occurs in two specific regions of the sperm head, prior to the initiation of the AR: the lower acrosome and the septum. Following the initiation of the AR, the cortical F-actin (upper acrosome) is lost as a result of the formation hybrid vesicles and not as a result of active depolymerization. Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

(CAP) was supplemented with 5 mg/ml bovine serum albumin (BSA) and (1 mg/ml)-coated coverslips to allow recordings. Unattached spermatozoa 24 mM NaHCO3, whereas the non-capacitating condition (NC) lacked were removed by gentle washing, and the chamber was filled with the NaHCO3. The pH was maintained at 7.4 for both cases. recording medium (NC medium lacking BSA) containing 100 nM SiR- actin. Recordings were performed at 37°C using a temperature controller CASA (OKO-TOUC, Okolab Inc., USA). Sperm were viewed with a Zeiss Aliquots of 3 μl of the sperm suspension were placed into a Leja slide with Observer Z.1 inverted microscope (Yokogawa spinning disc confocal) and 20 µm chamber depth pre-warmed to 37°C. The total motility of sperm an oil-immersion fluorescence objective (Zeiss plan Apocromat TIRF DIC loaded with 100 nM SiR-actin or vehicle (DMSO) was measured using the H/N2 63×/1.4 NA, oil DIC). A pre-centered fiber illuminator (Intelligent Sperm Class Analyzer (SCA) software (MICROPTIC S.L., Barcelona, Imaging Innovations, Inc.) was used as the light source. For collection Spain). The settings used for the analysis were as follows: frames acquired, excitation and emission SiR-actin fluorescence, a 640 nm laser and a Cy5 30; frame rate, 60 Hz; minimum area, 30 µm2; maximum area, 170 µm2; filter were used. Fluorescence images were acquired with an Andor Ixon 3 VCL or VAP, VCL; for VCL: statics <10 µm/s, slow-medium 15 µm/s, fast EMCCD camera, model DU-897E-CS0-#BV (Andor Technology) under >45 µm/s; progressive >50% of STR; points VAP 5; connectivity 18. At protocols written in SlideBook software version 6. For these experiments, least 300 cells were analyzed. we recorded one image every 5 s with an exposure time of 100 ms. An image of the plane was taken every 0.27 μm with a total of 20 planes Evaluation of F-actin levels by monitoring SiR-actin considering an approximate sperm depth of 5 μminthez plane; 100 fields fluorescence using image-based flow cytometry were acquired simultaneously, using an autofocus defined in a range of Sperm were incubated with 100 nM SiR-actin during 10 min in NC 10 μm in the xy plane. The images were obtained offline by generating the medium. Once loaded, sperm were incubated for 60 min in the appropriate maximum z-projection of the image stacks. As a control, we also used sperm medium (CAP or NC). The concentration of SiR-actin in the medium was from Lifeact-EGFP mice. For these experiments, a Nikon Eclipse Ti-E 100 nM during the whole experiment in order to get a constant signal and to inverted confocal microscope was used with an iXon Ultra 897 camera. avoid the probe interfering with actin dynamics. To evaluate F-actin levels, the SiR-actin loaded sperm were analyzed on an image-based flow In vivo super-resolution microscopy of F-actin in cytometer (Image Stream Mark II, Amnis, Seattle, WA). To determine single mouse sperm sperm viability, 100 nM of propidium iodide (PI) was added to the sperm Sperm live imaging was performed using the sperm loaded with SiR-actin as suspension. 488 nm and 642 nm solid-state lasers were used for excitation of described above. PI and SiR-actin, respectively. To reduce auto-fluorescence of unstained Super-resolution imaging measurements were performed on an Olympus cells, the power of the lasers was adjusted to 50 mW (488 nm) and 150 mW IX-81 inverted microscope configured for total internal reflection (642 nm). The instrument and INSPIRE software were set up as follows: fluorescence (TIRF) excitation (cellTIRF Illuminator, Olympus). The channels 01 and 09 for Brightfield, channel 05 for PI and channel 11 for excitation angle was set up such that the evanescence field had a SiR-actin. A 60× magnification was used. The flow rate was set to low penetration depth of ∼500 nm (Xcellence software v1.2, Olympus Soft speed/high sensitivity and stream alignment was adjusted when necessary. Imaging Solution GMBH). The samples were illuminated stroboscopically During acquisition, an area and aspect ratio thresholds (≥50–≤300 µm2, and using excitation sources depending on the fluorophore used. FM4-64 and ≤0.3, respectively) were set to acquire events compatible with sperm SiR-actin were excited with 568 or 640 nm diode-pumped solid-state lasers, morphology. Only single cells, excluding cellular aggregates, or debris were respectively. The maximum laser power, measured at the back aperture of analyzed. Additionally, a focus threshold for the exclusion of out of focus the objective lens, ranged between 20 to 25 mW, depending on the laser line events [gradient root mean square (gradient RMS) for channel 01 of ≥60] used. Beam selection and modulation of laser intensities were controlled was held. An additional gating of alive cells was made. Within these via Xcellence software v.1.2. A full multiband laser cube set was used parameters, the acquisition rate was of ∼1000–2000 cells per minute to discriminate the selected light sources (LF 405/488/561/635 A-OMF, (PI-negative cells). A total of 10,000 cells were recorded for each condition. Bright Line; Semrock). Fluorescence was collected using an Olympus Sperm images were analyzed using the provided analysis software IDEAS UApo N 100×/1.49 NA oil-immersion objective lens with an extra 1.6× and the SiR-actin fluorescence intensity for each sperm was obtained. intermediate magnification lens. Images were acquired with an Andor Ixon 3 EMCCD camera model DU-897ECSO#BV (Andor Technology) Comparison between SiR-actin and Alexa-Fluor-488–phalloidin under protocols written in Xcellence software version 1.2 (Olympus soft staining imaging solutions). To compare the staining of SiR-actin with the more widely used phalloidin, a standard phalloidin staining protocol was used as previously described 3B analysis (Romarowski et al., 2015). Cells were fixed in 0.1% glutaraldehyde and Sub-diffraction images were derived from the Bayesian analysis of the 1.5% formaldehyde in PBS for 1 h and collected by centrifugation at 1300 g stochastic Blinking and Bleaching (termed 3B analysis). Each super- for 5 min. The sperm pellet was immediately re-suspended and incubated resolution image required 300 images limited by diffraction at an acquisition with 50 mM NH4Cl in PBS for 15 min and washed twice by resuspension/ rate of 1 image every 61 ms, which involved obtaining a super-resolution centrifugation in PBS and once in distilled water. Water re-suspended cells image every 18.3 s. were used to prepare smears, which were air dried at room temperature Each of the image sequences was fed into the 3B microscopy analysis overnight. Smears were rinsed with PBS for 7 min then permeabilized using plugin of ImageJ (Cox et al., 2012), with a pixel size of 100 nm and a full- acetone at −20°C for 7 min, and washed three times in PBS. Slides were width half maximum of the point spread function of 290 nm (for FM4-64) incubated with 2 units of Alexa-Fluor-488–phalloidin in 100 µl of PBS and and 200 nm (for SiR-actin), both measured experimentally with 0.17 µm 100 nM of SiR-actin, under glass coverslips for 1 h at room temperature in fluorescent beads (PS-Speck microscope point source kit; Molecular humid conditions in the dark. Smears were washed three times with PBS, Probes, Inc.). All other parameters were set up as the default values. The once in distilled water and air dried at room temperature. For observation, 3B analysis was run in parallel over 200 iterations as recommended, and the they were mounted under glass coverslips using Citifluor AF1. Slides were final super-resolution reconstructions were created at a pixel size of 10 nm. examined using a Yokogawa spinning disk confocal microscope (Zeiss The spatial resolution observed in our imaging setup by 3B analysis Observer Z.1) and images were captured at 63×/1.4 NA (oil). was ∼50 nm.

Live imaging of F-actin in single mouse sperm SRRF For F-actin visualization in live cells, motile sperm were incubated with For each super-resolution reconstruction, five serial stacks were acquired 100 nM SiR-actin for 10 min in NC medium and then incubated for 60 min within an evanescent field of 500 nm, with an axial z spacing of 100 nm. in the appropriate medium, depending on the experiment performed, Each serial stack, composed of 300 temporal images collected with an containing 100 nM SiR-actin. Sperm were immobilized on concanavalin A exposure time of 5 to 20 ms at 10 Hz, was reconstructed with NanoJ-SRRF Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958 plugins of ImageJ (Gustafsson et al., 2016). For the analysis, we used the IX81 TIRF microscope. An oil immersion fluorescence objective (60×/1.45 following parameters: Annular radius: 0.5, Radiality magnification: 10×, NA) was used. During all the experiments, 16-bit images were obtained and Annular axis: 8. For the temporal analysis, we used the Temporal Radiality movies were processed and analyzed with macros written in ImageJ Auto-Correlations algorithm of 2nd order, with temporal integrated (National Institutes of Health). Regions of interest were drawn on each sperm correlation and intensity ponderation. All other parameters were set up as in the movie for quantification. A plot of the fluorescence intensity of the default options. The radiality maps were drift corrected using pre- each spermatozoon versus time was generated in Origin 6.0 (OriginLab calculated drift tables obtained with the Estimate Drift tool of NanoJ-SRRF, Corporation). Fluorescence is expressed as (F−F0)/F0, where F0 is the considering a time averaging of 300 images. average fluorescence intensity value of the frames before the addition of ionomycin. When brightness and contrast were adjusted, this was done STORM equally in all images or movies filmed under the same conditions. Because SiR-actin is not suitable for STORM, phalloidin was used as described below. Sperm samples were seeded in poly-lysinated coverslips Statistical analysis (Corning #1.5) and air dried for 5 min. Then, samples were fixed and Data are expressed as mean±s.e.m. or ±s.d. for at least three experiments for permeabilized with 0.3% (v/v) glutaraldehyde and 0.25% (v/v) Triton all determinations. Statistical analyses were performed with GraphPad X-100 in cytoskeleton buffer [CB; MES (10 mM, pH6.1), NaCl (150 mM), Prism version 4.0 for Windows, GraphPad Software (San Diego, CA) or by EGTA (5 mM), glucose (5 mM), and MgCl2 (5 mM)] for 1 min. Cells were using the R language environment [R 3.3.3 GUI 1.69 Mavericks build washed with CB three times for 5 min each, and then incubated with 2% (7328)]. The statistical analysis performed is indicated in the corresponding (v/v) glutaraldehyde in CB for 15 min. Then cells were then washed with figure legend. P<0.05 was considered statistically significant. CB twice for 10 min each. To avoid background fluorescence caused by glutaraldehyde fixation, samples were incubated with 0.1% (w/v) of sodium Acknowledgements borohydride in PBS for 7 min. After that, samples were washed with PBS We thank Santiago Di Pietro (Department of Biochemistry and Molecular Biology, twice for 5 min each, and incubated with 0.5 μM Alexa-Fluor-647– Colorado State University, USA) for providing Lifeact-EGFP mice. We also would like phalloidin in PBS for 1 h. Cells were then washed with PBS three times to thank Dr James Bamburg for the helpful discussion about this project. We also for 5 min each and immediately mounted in STORM imaging buffer. thank Lis Puga Molina and Jose Luis De La Vega-Beltran for their insightful ́ Images were acquired using Andor IQ 2.3 software in a custom-built comments. We are also thankful to Arturo Pimentel, Andres Saralegui and Xochitl microscope equipped with an Olympus PlanApo 100×/1.45 NA objective Alvarado from LNMA-UNAM for their helpful discussions and assistance with the microscopes. Microscopy equipment was provided and maintained through Consejo and a CRISP ASI autofocus system (Weigel et al., 2011). Alexa Fluor 647 Nacional de Ciencia y Tecnologıá (CONACYT) grants 123007, 232708, 260541, was excited with a 640 nm laser (DL640-150-O, CrystaLaser, Reno, NV) 280487 and 293624. under continuous illumination. Initially, the photo-switching rate was sufficient to provide a substantial fluorophore density. However, as Competing interests fluorophores irreversibly photo-bleached, a 405 nm laser was introduced The authors declare no competing or financial interests. to enhance photo-switching. The intensity of the 405 nm laser was adjusted in the range of 0.01–0.5 mW to maintain an appropriate density of active Author contributions fluorophores. A cylindrical lens with a focal lens of 1 m was placed in the Conceptualization: P.E.V., Dario Krapf, A.G., A.D., M.G.B.; Methodology: P.T.R., detection path in order to achieve a 3D resolution (Huang et al., 2008). M.G.G., X.X., G.C.-J., C.S.-C., A.G.; Software: Diego Krapf, A.G.; Validation: A calibration curve for axial localization was generated with 50 nm gold G.M.L., H.V.R.-G., A.G.; Formal analysis: A.R., Diego Krapf; Investigation: A.R., nanoparticles (Nanopartz, Loveland, CO) immobilized on a coverslip. A.G.V.F., P.T.R., M.G.G., G.M.L., H.V.R.-G.; Resources: C.S.-C., P.E.V., A.G., A.D.; The images were acquired by a water-cooled, back-illuminated EMCCD Data curation: A.G.V.F.; Writing - original draft: A.R., A.D., M.G.B.; Writing - review & camera (Andor iXon DU-888) operated at −85°C at a rate of 23 frames/s; editing: Diego Krapf, P.E.V., Dario Krapf, A.G., A.D., M.G.B.; Visualization: X.X., 50,000 frames were collected to generate a super-resolution image. Super- C.S.-C., Diego Krapf, Dario Krapf; Supervision: A.D., M.G.B.; Project administration: resolution image reconstruction, single-molecule localization, and drift M.G.B.; Funding acquisition: M.G.B. correction using image cross correlation and reconstruction were all performed with ThunderSTORM plugin in ImageJ (Ovesný et al., 2014). Funding This work was supported by the National Institutes of Health (RO1 TW008662 to M.G.B.), the Eunice Kennedy Shriver National Institute of Child Health and Human TIRF microscopy to visualize cortical F-actin and plasma Development, NIH (RO1 HD38082 to P.E.V.), Agencia Nacional de Promoción membrane in single mouse sperm Cientıficá y Tecnológica (PICT 2015-2294 to M.G.B.), National Science Foundation Recordings were performed using the sperm loaded with SiR-actin as (1401432 to D.K.). PAPIIT-UNAM, IN205516; Consejo Nacional de Ciencia y described above, but in this case the recording medium contained 1 μM Tecnologıá (CONACyT) (CB 2015/255914, 280478, 252969 and 253952 to A.D.). FM4-64. For these experiments, the inverted Olympus IX81 TIRF A.G. thanks CONACyT (252213) and DGAPA-PAPIIT (No. IA202417). We also microscope equipped with an Andor IXON 3 camera was used, as acknowledge the Rene Baron, Fortabat and Williams Foundations, Programa de Movilidad en el Posgrado de la Red de Macro Universidades Públicas de América explained in previous sections. Fluorescence was collected using an Latina y el Caribe for funding support. Deposited in PMC for release after Olympus UApo N 100×/1.49 NA oil-immersion objective lens with an extra 12 months. 1.6× intermediate magnification lens. For SiR-actin excitation, a 640 nm laser was used and, for FM4-64 excitation, a 491 nm laser was used. The Supplementary information emission filter window of 670–700 nm was used. The delay between one Supplementary information available online at channel and the other was of 30 ms. The diffraction-limited images obtained http://jcs.biologists.org/lookup/doi/10.1242/jcs.218958.supplemental by TIRF microscopy were used for super-resolution reconstruction as explained above. References Baltiérrez-Hoyos, R., Roa-Espitia, A. L. and Hernández-González, E. O. (2012). The association between CDC42 and caveolin-1 is involved in the regulation of Live imaging of F-actin dynamics and acrosomal exocytosis in capacitation and acrosome reaction of guinea pig and mouse sperm. single mouse sperm Reproduction 144, 123-134. Recordings were performed using capacitated sperm loaded with SiR-actin Belmonte, S. A., Mayorga, L. S. and Tomes, C. N. (2016). The molecules of sperm as described above, but in this case the recording medium contained 1 μM exocytosis. Adv. Anat. Embryol. Cell Biol. 220, 71-92. FM4-64. Sperm were recorded over time, before and after the addition Branham, M. T., Mayorga, L. S. and Tomes, C. N. (2006). Calcium-induced μ acrosomal exocytosis requires cAMP acting through a protein kinase of 10 M ionomycin or 100 µM progesterone. SiR-actin and FM4-64 A-independent, Epac-mediated pathway. J. Biol. Chem. 281, 8656-8666. fluorescence acquisitions were performed as described above. Two images Branham, M. T., Bustos, M. A., De Blas, G. A., Rehmann, H., Zarelli, V. E. P., per second were acquired for periods of 20 min, using the inverted Olympus Treviño, C. L., Darszon, A., Mayorga, L. S. and Tomes, C. N. (2009). Epac Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

activates the small G proteins Rap1 and Rab3A to achieve exocytosis. J. Biol. Inoue, N., Ikawa, M., Isotani, A. and Okabe, M. (2005). The immunoglobulin Chem. 284, 24825-24839. superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434, Brener, E., Rubinstein, S., Cohen, G., Shternall, K., Rivlin, J. and Breitbart, H. 234-238. (2003). Remodeling of the actin cytoskeleton during mammalian sperm Jones, R., James, P. S., Howes, L., Bruckbauer, A. and Klenerman, D. (2007). capacitation and acrosome reaction. Biol. Reprod. 68, 837-845. Supramolecular organization of the sperm plasma membrane during maturation Buffone, M. G., Hirohashi, N. and Gerton, G. L. (2014). Unresolved questions and capacitation. Asian J. Androl. 9, 438-444. concerning mammalian sperm acrosomal exocytosis. Biol. Reprod. 90, 112. Korley, R., Pouresmaeili, F. and Oko, R. (1997). Analysis of the protein Chowdhury, H. H., Popoff, M. R. and Zorec, R. (1999). Actin cytoskeleton composition of the mouse sperm perinuclear theca and characterization of its depolymerization with clostridium spiroforme toxin enhances the secretory activity major protein constituent. Biol. Reprod. 57, 1426-1432. of rat melanotrophs. J. Physiol. 521, 389-395. La Spina, F. A., Puga Molina, L. C., Romarowski, A., Vitale, A. M., Falzone, T. L., Chung, J.-J., Shim, S.-H., Everley, R. A., Gygi, S. P., Zhuang, X. and Clapham, Krapf, D., Hirohashi, N. and Buffone, M. G. (2016). Mouse sperm begin to D. E. (2014). Structurally distinct Ca2+ signaling domains of sperm flagella undergo acrosomal exocytosis in the upper isthmus of the oviduct. Dev. Biol. 411, orchestrate tyrosine phosphorylation and motility. Cell 157, 808-822. 172-182. Chung, J.-J., Miki, K., Kim, D., Shim, S.-H., Shi, H. F., Hwang, J. Y., Cai, X., Iseri, Lucchesi, O., Ruete, M. C., Bustos, M. A., Quevedo, M. F. and Tomes, C. N. Y., Zhuang, X. and Clapham, D. E. (2017). CatSperζ regulates the structural (2016). The signaling module cAMP/Epac/Rap1/PLCε/IP3 mobilizes acrosomal continuity of sperm Ca2+ signaling domains and is required for normal fertility. calcium during sperm exocytosis. Biochim. Biophys. Acta Mol. Cell Res. 1863, eLife 6, e23082. 544-561. Clermont, Y., Oko, R. and Hermo, L. (1990). Immunocytochemical localization of Lukinavičius, G., Reymond, L., D’Este, E., Masharina, A., Göttfert, F., Ta, H., proteins utilized in the formation of outer dense fibers and fibrous sheath in rat Güther, A., Fournier, M., Rizzo, S., Waldmann, H. et al. (2014). Fluorogenic spermatids: an electron microscope study. Anat. Rec. 227, 447-457. probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731-733. Cohen, G., Rubinstein, S., Gur, Y. and Breitbart, H. (2004). Crosstalk between Magliocca, V., Petrini, S., Franchin, T., Borghi, R., Niceforo, A., Abbaszadeh, Z., protein kinase A and C regulates phospholipase D and F-actin formation during Bertini, E. and Compagnucci, C. (2017). Identifying the dynamics of actin and sperm capacitation. Dev. Biol. 267, 230-241. tubulin polymerization in iPSCs and in iPSC-derived neurons. Oncotarget 8, Cooper, J. A. (1987). Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 111096-111109. 105, 1473-1478. Mata-Martınez,́ E., Darszon, A. and Treviño, C. L. (2018). pH-dependent Ca +2 Cox, S., Rosten, E., Monypenny, J., Jovanovic-Talisman, T., Burnette, D. T., oscillations prevent untimely acrosome reaction in human sperm. Biochem. Lippincott-Schwartz, J., Jones, G. E. and Heintzmann, R. (2012). Bayesian Biophys. Res. Commun. 497, 146-152. localization microscopy reveals nanoscale podosome dynamics. Nat. Methods Melak, M., Plessner, M. and Grosse, R. (2017). Actin visualization at a glance. 9, 195-200. J. Cell Sci. 130, 525-530. Dan, J. C. (1952). Studies on the acrosome. I. Reaction to egg-water. Biol. Bull. Miranda, P. V., Allaire, A., Sosnik, J. and Visconti, P. E. (2009). Localization of 103, 54-66. low-density detergent-resistant membrane proteins in intact and acrosome- De Blas, G. A., Roggero, C. M., Tomes, C. N. and Mayorga, L. S. (2005). reacted mouse sperm. Biol. Reprod. 80, 897-904. Dynamics of SNARE assembly and disassembly during sperm acrosomal Muallem, S., Kwiatkowska, K., Xu, X. and Yin, H. L. (1995). Actin filament exocytosis. PLoS Biol. 3, e323. disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J. Cell De Jonge, C. J., Han, H.-L., Lawrie, H., Mack, S. R. and Zaneveld, L. J. D. (1991). Biol. 128, 589-598. Modulation of the human sperm acrosome reaction by effectors of the adenylate Muro, Y., Hasuwa, H., Isotani, A., Miyata, H., Yamagata, K., Ikawa, M., cyclase/cyclic AMP second-messenger pathway. J. Exp. Zool. 258, 113-125. Yanagimachi, R. and Okabe, M. (2016). Behavior of mouse spermatozoa in De La Vega-Beltran, J. L., Sánchez-Cárdenas, C., Krapf, D., Hernandez- the female reproductive tract from soon after mating to the beginning of González, E. O., Wertheimer, E., Treviño, C. L., Visconti, P. E. and Darszon, A. fertilization. Biol. Reprod. 94, 80. (2012). Mouse sperm membrane potential hyperpolarization is necessary and Oko, R. and Morales, C. R. (1994). A novel testicular protein, with sequence sufficient to prepare sperm for the acrosome reaction. J. Biol. Chem. 287, similarities to a family of lipid binding proteins, is a major component of the rat 44384-44393. sperm perinuclear Theca. Dev. Biol. 166, 235-245. D’Este, E., Kamin, D., Göttfert, F., El-Hady, A. and Hell, S. W. (2015). STED Ovesný, M., Křıź̌ek, P., Borkovec, J., Švindrych, Z. and Hagen, G. M. (2014). nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data neurons. Cell Rep. 10, 1246-1251. analysis and super-resolution imaging. Bioinformatics 30, 2389-2390. Ehre, C., Rossi, A. H., Abdullah, L. H., De Pestel, K., Hill, S., Olsen, J. C. and Pelletán, L. E., Suhaiman, L., Vaquer, C. C., Bustos, M. A., De Blas, G. A., Vitale, Davis, C. W. (2004). Barrier role of actin filaments in regulated mucin secretion N., Mayorga, L. S. and Belmonte, S. A. (2015). ADP ribosylation factor 6 (ARF6) from airway goblet cells. Am. J. Physiol. Cell Physiol. 288, C46-C56. promotes acrosomal exocytosis by modulating lipid turnover and Rab3A Fiedler, S. E., Bajpai, M. and Carr, D. W. (2008). Identification and characterization activation. J. Biol. Chem. 290, 9823-9841. of RHOA-interacting proteins in bovine spermatozoa. Biol. Reprod. 78, 184-192. Pelletier, R.-M., Trifaro, J.-M., Carbajal, M. E., Okawara, Y. and Vitale, M. L. Finkelstein, M., Etkovitz, N. and Breitbart, H. (2010). Role and regulation of sperm (1999). Calcium-dependent actin filament-severing protein scinderin levels and gelsolin prior to fertilization. J. Biol. Chem. 285, 39702-39709. localization in bovine testis, epididymis, and spermatozoa. Biol. Reprod. 60, Fukami, K., Nakao, K., Inoue, T., Kataoka, Y., Kurokawa, M., Fissore, R. A., 1128-1136. Nakamura, K., Katsuki, M., Mikoshiba, K., Yoshida, N. et al. (2001). Puga Molina, L. C., Luque, G. M., Balestrini, P. A., Marın-Briggiler,́ C. I., Requirement of phospholipase Cdelta4 for the zona pellucida-induced Romarowski, A. and Buffone, M. G. (2018). Molecular basis of human sperm acrosome reaction. Science 292, 920-923. capacitation. Front. Cell Dev. Biol. 6, 72. Fukami, K., Yoshida, M., Inoue, T., Kurokawa, M., Fissore, R. A., Yoshida, N., Quevedo, M. F., Lucchesi, O., Bustos, M. A., Pocognoni, C. A., De la Iglesia, Mikoshiba, K. and Takenawa, T. (2003). Phospholipase Cδ4 is required for Ca 2+ P. X. and Tomes, C. N. (2016). The Rab3A-22A chimera prevents sperm mobilization essential for acrosome reaction in sperm. J. Cell Biol. 161,79-88. exocytosis by stabilizing open fusion pores. J. Biol. Chem. 291, 23101-23111. Gasman, S., Chasserot-Golaz, S., Malacombe, M., Way, M. and Bader, M.-F. Riedl, J., Flynn, K. C., Raducanu, A., Gärtner, F., Beck, G., Bösl, M., Bradke, F., (2004). Regulated exocytosis in neuroendocrine cells: a role for Massberg, S., Aszodi, A., Sixt, M. et al. (2010). Lifeact mice for studying F-actin subplasmalemmal Cdc42/N-WASP–induced actin filaments. Mol. Biol. Cell 15, dynamics. Nat. Methods 7, 168-169. 520-531. Romarowski, A., Battistone, M. A., La Spina, F. A., Puga Molina, L. D. C., Luque, Gervasi, M. G., Xu, X., Carbajal-Gonzalez, B., Buffone, M. G., Visconti, P. E. and G. M., Vitale, A. M., Cuasnicu, P. S., Visconti, P. E., Krapf, D. and Buffone, Krapf, D. (2018). The actin cytoskeleton of the mouse sperm flagellum is M. G. (2015). PKA-dependent phosphorylation of LIMK1 and Cofilin is essential organized in a helical structure. J. Cell Sci. 131, jcs.215897. for mouse sperm acrosomal exocytosis. Dev. Biol. 405, 237-249. Gustafsson, N., Culley, S., Ashdown, G., Owen, D. M., Pereira, P. M. and Romarowski, A., Sánchez-Cárdenas, C., Ramırez-Gó ́mez, H. V., Puga Molina, Henriques, R. (2016). Fast live-cell conventional fluorophore nanoscopy with L. D. C., Treviño, C. L., Hernández-Cruz, A., Darszon, A. I. and Buffone, M. G. ImageJ through super-resolution radial fluctuations. Nat. Commun. 7, 12471. (2016a). A specific transitory increase in intracellular calcium induced by Hino, T., Muro, Y., Tamura-Nakano, M., Okabe, M., Tateno, H. and Yanagimachi, progesterone promotes acrosomal exocytosis in mouse sperm. Biol. Reprod. R. (2016). The behavior and acrosomal status of mouse spermatozoa in vitro, and 94, 1-12. within the oviduct during fertilization after natural mating. Biol. Reprod. 95, 50-50. Romarowski, A., Luque, G. M., La Spina, F. A., Krapf, D. and Buffone, M. G. Hirohashi, N., La Spina, F. A., Romarowski, A. and Buffone, M. G. (2015). (2016b). Role of actin cytoskeleton during mammalian sperm acrosomal Redistribution of the intra-acrosomal EGFP before acrosomal exocytosis in exocytosis. Adv. Anat. Embryol. Cell Biol. 220, 129-144. mouse spermatozoa. Reproduction 149, 657-663. Sánchez-Cárdenas, C., Servın-Vences,́ M. R., José, O., Treviño, C. L., Huang, B., Wang, W., Bates, M. and Zhuang, X. (2008). Three-dimensional super- Hernández-Cruz, A. and Darszon, A. (2014). Acrosome reaction and Ca2+ resolution imaging by stochastic optical reconstruction microscopy. Science imaging in single human spermatozoa: new regulatory roles of [Ca2+]i. Biol.

319, 810-813. Reprod. 91, 67. Journal of Cell Science

13 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958

Satouh, Y., Inoue, N., Ikawa, M. and Okabe, M. (2012). Visualization of the moment Suzuki, K. G. N., Ando, H., Komura, N., Fujiwara, T. K., Kiso, M. and Kusumi, A. of mouse sperm-egg fusion and dynamic localization of IZUMO1. J. Cell Sci. 125, (2017). Development of new ganglioside probes and unraveling of raft domain 4985-4990. structure by single-molecule imaging. Biochim. Biophys. Acta 1861, 2494-2506. Selvaraj, V., Asano, A., Buttke, D. E., Sengupta, P., Weiss, R. S. and Travis, A. J. Torregrosa-Hetland, C. J., Villanueva, J., Giner, D., Lopez-Font, I., Nadal, A., Quesada, I., Viniegra, S., Exposito-Romero, G., Gil, A., Gonzalez-Velez, V. (2009). Mechanisms underlying the micron-scale segregation of sterols and G M1 et al. (2011). The F-actin cortical network is a major factor influencing the in live mammalian sperm. J. Cell. Physiol. 218, 522-536. organization of the secretory machinery in chromaffin cells. J. Cell Sci. 124, Sosnik, J., Miranda, P. V., Spiridonov, N. A., Yoon, S.-Y., Fissore, R. A., 727-734. Johnson, G. R. and Visconti, P. E. (2009). Tssk6 is required for Izumo Vandekerckhove, J., Deboben, A., Nassal, M. and Wieland, T. (1985). The relocalization and gamete fusion in the mouse. J. Cell Sci. 122, 2741-2749. phalloidin binding site of F-actin. EMBO J. 4, 2815-2818. Sosnik, J., Buffone, M. G. and Visconti, P. E. (2010). Analysis of CAPZA3 Visegrády, B., Lőrinczy, D., Hild, G., Somogyi, B. and Nyitrai, M. (2004). The localization reveals temporally discrete events during the acrosome reaction. effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin J. Cell. Physiol. 224, 575-580. filaments. FEBS Lett. 565, 163-166. Spungin, B., Margalit, I. and Breitbart, H. (1995). Sperm exocytosis reconstructed Weigel, A. V., Simon, B., Tamkun, M. M. and Krapf, D. (2011). Ergodic and in a cell-free system: evidence for the involvement of phospholipase C and actin nonergodic processes coexist in the plasma membrane as observed by single- molecule tracking. Proc. Natl. Acad. Sci. USA 108, 6438-6443. filaments in membrane fusion. J. Cell Sci. 108, 2525-2535. Yamazaki, S., Harata, M., Idehara, T., Konagaya, K., Yokoyama, G., Hoshina, H. Stival, C., La Spina, F. A., BaróGraf, C., Arcelay, E., Arranz, S. E., Ferreira, J. J., and Ogawa, Y. (2018). Actin polymerization is activated by terahertz irradiation. Le Grand, S., Dzikunu, V. A., Santi, C. M., Visconti, P. E. et al. (2015). Src Sci. Rep. 8, 9990. kinase is the connecting player between Protein Kinase A (PKA) activation and Zhou, C., Huang, L., Shi, D.-S. and Jiang, J.-R. (2017). Effects of latrunculin A on hyperpolarization through SLO3 potassium channel regulation in mouse sperm. the relocation of sperm IZUMO1 during gamete interaction in mouse. Mol. Reprod. J. Biol. Chem. 290, 18855-18864. Dev. 84, 1183-1190. Journal of Cell Science